Energy transfer between Trp 59 in horse cytochrome c and the heme provides a convenient probe for observing the collapse of the polypeptide chain during folding. Previous kinetic folding studies , based on steady state fluorescence, absorbance and amide protection revealed a complex folding mechanism with several parallel pathways and evidence for a compact intermediate formed in the sub-millisecond time scale. Fluorescence lifetime measurements at early stages of the folding promise to provide valuable information on the overall chain conformation in early intermediates. In a previous attempt we found that this can be done quite simply by interfacing a continues flow mixing apparatus with the picosecond photon-counting instrument. The results were technically encouraging, but under the refolding conditions chosen, we detected only unfolded species, whereas any partially of fully folded molecules showed lifetimes less than the 30 ps instrument response. We will attempt the experiment again under several different refolding conditions (pH and final denaturant concentrations) known to affect the folding mechanism. For example, at pH 5 cytochrome c follows a more direct folding pathway than at pH 7, and at pH 2, the protein forms a compact intermediate (A-state) which we previously characterized by time resolved fluorescence.